CN106501535B - A kind of kit of the compound releasing agent of ANS salt and detection Blood cortisol - Google Patents
A kind of kit of the compound releasing agent of ANS salt and detection Blood cortisol Download PDFInfo
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- CN106501535B CN106501535B CN201611082658.7A CN201611082658A CN106501535B CN 106501535 B CN106501535 B CN 106501535B CN 201611082658 A CN201611082658 A CN 201611082658A CN 106501535 B CN106501535 B CN 106501535B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The present invention discloses a kind of kit of the compound releasing agent of ANS salt and detection Blood cortisol, and kit includes following components：It is coated with the magnetic bead of cortisol antibody, the cortisol BSA compounds of acridinium ester label, the compound releasing agent of ANS salt and cortisol calibration object.The cortisol of combining form in blood of human body sample is changed into the cortisol of free form by the compound releasing agent of ANS salt of the present invention, and the content of free cortisol is detected by acridinium ester chemiluminescent.The compound releasing agent of ANS salt can be combined with different glucocorticosteroid albumen, can fully release the cortisol of reference state in blood.Acridinium ester chemiluminescent shines for flash type, and when illuminating is few, being capable of photon caused by catching reaction completely；Acridinium ester is artificial synthetic, higher to the anti-interference of sample interfering substance, and the content that free cortisol is detected using acridinium ester label is very sensitive, keeps the assay of total cortisol in blood sample more accurate.
Description
Technical field
The present invention relates to technical field of immunoassay more particularly to a kind of compound releasing agents of ANS salt and skin in detection blood
The kit of matter alcohol.
Background technology
Cortisol (Cortisol) is also known as " hydrocortisone ", is a kind of sugared cortex that adrenal gland generates in stress reaction
Hormone.When people takes in cholesterol from food, under the action of body generates stress reaction, cholesterol is via in adrenal gland
" glucocorticoid system " becomes cortisol, and is secreted into blood in a free form.Cortisol, which has, adjusts carbon hydrate
The function of object metabolism；Immunosupress and anti-inflammatory effect；To hypothalamus secretion corticoliberim and hang down
The corticotropin of body secretion plays negative-feedback regu- lation.Therefore, by detecting the content of cortisol, kidney can be diagnosed
The functional status of upper gland, while can be with the functional status of indirect observation hypophysis.
Due to there are a large amount of corticosteroid globulin and albumin in blood of human body can be with the cortex dissociated in blood
Alcohol combines, and cortisol in conjunction with after cannot be from glomerular filtration, and cortisol just loses bioactivity at this time.And it is only a small amount of
Cortisol exists in a free form, and free cortisol can be filtered out by glomerulus, into urine.Therefore want accurate
The content of cortisol in blood sample is measured, how to release free cortisol is just particularly important.
The releasing agent selected on the market at present is mainly danazol, and structural formula is as follows：
Danazol (Danazol) is a kind of artificial synthesized steroidal heterocyclic compound, i.e. androgenous 11 7a- Ethisterones
Derivative, for white or creamy white crystals or crystalline powder.Due to not soluble in water, it is necessary to pass through organic solvent, such as DMF
The dissolvings such as (dimethylformamide), chloroform, acetone.However these organic solvents can influence cortisol release, so as to cause blood
In dissociate Determination of cortisol detection it is inaccurate.Therefore selection can not only be dissolved in water but also play the releasing agent of cortisol release action
It is particularly important.
The method of current cortisol immunodiagnosis is mainly the following method：
1. different luminol and its Direct Electrochemistry of derivative substance markers shine
Principle：It uses different luminol and its derivative as luminous substrate, reaction speed is improved using the catalyst of metal ion
Degree and sensitivity, use NaOH+H2O2Make the exciting liquid that shines, the direct chemiluminescence of flash type is acquired entire using photon counter
The luminous measurement method counted of reaction process.
It is insufficient：Different luminol and its derivative shine as flash type, and luminous intensity depends in immune response
The concentration of enzyme, if insufficient without using catalyst or catalytic amount, luminous signal dies down, and the amount of catalyst is difficult control；Its
Secondary, haemolysis interference of the different luminol in by sample influences the measurement of free Determination of cortisol in blood.
2. electrochemical luminescence
Principle：ECLI method luminescence reagent markers are the tris (bipyridine) ruthenium [Ru (bpy) of divalent3]2+, another reagent is 3 third
Amine TPA can lose electronics simultaneously in anode surface both the above electroactive substance and be aoxidized.The tripropyl amine (TPA) of oxidation loses
One H+And become strong reductant, by the tris (bipyridine) ruthenium [Ru (bpy) of oxidized form3]3+It is reduced to [the Ru (bpy) of excitation state3
]2+, discharge photon immediately and restore the luminous substrate of ground state, this process is carried out in electrode surface, constantly sent out again and again
Photon and often keep the constant of concentration of substrate.
It is insufficient：It is excessively sensitive to environmental factor and other nonspecific reactions；For metal ion, EDTA or other anti-freezings
Agent, the metallo-chelate of preservative are excessively sensitive；The deposition of reaction by-product is also a potential interference source；It is to exceeding pH
The sample of 6-7 is very sensitive；O2In the case where voltage conditions are added, shine under the conditions of 620nm, electrochemical luminescence is in by reaction system
The interference of dissolved oxygen；Wiper mechanism flow cell cleans, and flow cell is recycled, higher for cleaning requirement.
3. luminol-HRP systems
Principle：Luminol is used under alkaline condition, to form a pairs of anion as luminous substrate.This pairs of anion can quilt
Dioxygen oxidation generates a highly unstable organic peroxide.The peroxide is very unstable, decomposites nitrogen immediately, raw
At the 3- aminophthalic acids of triplet state (excitation state).When the latter is restored to ground state, launch photon, about 20 points of continuous illumination
Clock is longer.
It is insufficient：HRP heavy metal ion, temperature, NaN in by sample3Etc. factors interference；Reagent stability is by sample pilot scale
Agent stability is restricted by HRP stability；Marker is protein molecular, and marker addition increases antigen-antibody reaction steric hindrance.
4. adamantane-ALP systems
Principle：Use spiral adamantane to issue unboiled water solution in alkaline phosphatase enzyme effect as luminous substrate, obtain one it is medium
Stable intermediate A MPD-(2~30 minutes half-life period).Electronics transfer is cracked into the adamantane of a molecule in this intermediate molecule
Ketone and a molecule are in oxybenzoic acid methyl esters anion between excitation state, the light of 470nm are generated when it returns to ground state, and continue
Dozens of minutes belongs to wide variety of glow-type enzyme-catalyzed chemical luminescence, and count measurement is carried out using photon counter interception one section fluorescent lifetime.
It is insufficient：The ALP factors such as heavy metal ion, temperature interference in by sample；Reagent stability is steady by reagent in sample
It is qualitative to be restricted by ALP stability；Marker is protein molecular, and marker addition increases antigen-antibody reaction steric hindrance.
It can be seen from the above, at present there is drawback in its clinical application of existing external diagnosis reagent.
Therefore, the existing technology needs to be improved and developed.
Invention content
In view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of compound releasing agents of ANS salt and detection
The kit of Blood cortisol, it is intended to solve the above problem existing for existing external diagnosis reagent.
Technical scheme is as follows：
A kind of kit of detection Blood cortisol, wherein including following components：The magnetic bead of coating cortisol antibody,
Cortisol BSA compounds, the compound releasing agent of ANS salt and the cortisol calibration object of acridinium ester label.
The kit of the detection Blood cortisol, wherein further include the cleaning solution A and luminous cleaning solution B that shines.
The kit of the detection Blood cortisol, wherein the magnetic bead be coated with carboxyl, epoxy group,
Any one in toluene Huang acyl group.
The kit of the detection Blood cortisol, wherein mole of acridinium ester and the compound substance markers of cortisol BSA
Ratio is between 1:2.5-1:Between 10.
The kit of the detection Blood cortisol, wherein the cortisol BSA compounds of the acridinium ester label are
Cortisol-3-BSA compounds.
The kit of the detection Blood cortisol, wherein the cortisol calibration object is the alcohol hemocrinia that decorticates
Clear diluted cortisol antigen.
The kit of the detection Blood cortisol, wherein the luminous cleaning solution A includes following components：50-
The Tween- of the disodium hydrogen phosphate of 100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L, 0.1%-10%
20, the nitric acid of the hydrogen peroxide of liquid bio preservative Proclin-300,0.5%-5% of 0.1%-10% and 1-2M.
The kit of the detection Blood cortisol, wherein the luminous cleaning solution B includes following components：50-
The Tween- of the disodium hydrogen phosphate of 100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L, 0.1%-10%
20, the sodium hydroxide and 0.1%-2% of liquid bio preservative Proclin-300,0.01-2.5M of 0.1%-10%
TritonX-100。
A kind of compound releasing agent of ANS salt, wherein the compound releasing agent of ANS salt includes following components：3-15mg/ml
ANS salt, 0.1-0.5%NaN3, 100-200mM NaCl and 20-60mM barbital sodiums.
The compound releasing agent of ANS salt, wherein the ANS salt has following structure：
Above-mentioned R+For NH4 +、K+Or Na+。
Advantageous effect：The present invention is turned the cortisol of combining form in blood of human body sample by the compound releasing agent of ANS salt
Become the cortisol of free form, and detects the content of free cortisol by chemiluminescent new technique.The ANS salt of selection
Compound releasing agent can be combined with different glucocorticosteroid albumen, can fully release the cortisol of reference state in blood
Come；The chemiluminescence is acridinium ester chemiluminescent, and acridinium ester chemiluminescent shines for flash type, and when illuminating is few, can
Photon caused by complete catching reaction；Acridinium ester is artificial synthetic, higher to the anti-interference of sample interfering substance, profit
The content that free cortisol is detected with acridinium ester label is very sensitive, make the assay of total cortisol in blood sample more subject to
Really.
Description of the drawings
Fig. 1 is the theoretical concentration of the embodiment of the present invention and actually detected concentration relationship schematic diagram.
Specific implementation mode
The present invention provides a kind of kit of the compound releasing agent of ANS salt and detection Blood cortisol, to make the present invention's
Purpose, technical solution and effect are clearer, clear, and the present invention is described in more detail below.It should be appreciated that this place is retouched
The specific embodiment stated is only used to explain the present invention, is not intended to limit the present invention.
The present invention provides a kind of compound releasing agent of ANS salt, wherein the compound releasing agent of ANS salt includes following components：3-
15mg/ml ANS salt, 0.1-0.5%NaN3, 100-200mM NaCl and 20-60mM barbital sodiums.
ANS salt (8-anilino-1-naphthalene sulfonic acid salt) is a kind of fluorescent dye, is had to the hydrophobic surface of protein high affine
Power.ANS salt can be attached to the protein surface of low polar region.It is larger that these attributes make ANS salt be highly suitable for affinity
Hydrophobic ligand and its corresponding binding protein.And cortisol be small molecule parahormone, usually in blood with binding protein
In conjunction with, and cortisol is combined not have bioactivity, free cortisol can be made to obtain adequately using addition ANS salt releasing agent
Release；Compared to traditional releasing agent danazol, ANS salt need not dissolve in organic solvent, can directly dissolve in water,
Operating process is reduced, reagent raw material consumables cost is reduced, and ANS salt is much larger than the practical release efficiency of free cortisol and reaches
That azoles effectively improves sensitivity and accuracy to its release efficiency.
Wherein, the structural formula of the barbital sodium is as follows,
Barbital sodium (BBTS) is a kind of chemical substance, and barbital sodium and albumen are interacted with electrostatic force, which is
The spontaneous supermolecular mechanism process that one entropy increase, BBTS free energys reduce.It is raw for the small molecules such as cortisol and protein etc.
Active force type between object macromolecular has hydrophobic force, hydrogen bond, Van der Waals force and electrostatic force.Under certain temperature and pressure, egg
The space subdomain of white macromolecular forms cylinder-like structure in such a way that notch is opposite, and almost all of hydrophobic amino acid is residual
Base is all embedded in cylinder interior, constitutes hydrophobic pocket, and there are a kind of electrostatic forces between BBTS and albumen so that BBTS molecules
It is in this columnar hydrophobic pocket with the binding site of albumen, the cortisol small molecule that part combines can be made to be released.
And ANS salt actions are in the larger hydrophobic ligand of affinity and its corresponding binding protein hydrophobic surface.When releasing ANS salt is compound
It puts and ANS salt and BBTS is added in agent, free cortisol in binding protein can be made more fully to be discharged.
The present invention also provides a kind of kit preferred embodiments of detection Blood cortisol, wherein including following components：
Be coated with the magnetic bead of cortisol antibody, the cortisol BSA compounds of acridinium ester label, the compound releasing agent of ANS salt as described above with
Cortisol calibration object.Preferably, the kit that the present invention detects Blood cortisol further includes shine cleaning solution A and the cleaning that shines
Liquid B.
Specifically, the present invention is coated with the magnetic bead of cortisol antibody, wherein the particle size of the magnetic bead is 1.5 μm of -2.8 μ
m.Preferably, the kernel of the magnetic bead is ferroso-ferric oxide, and the magnetic bead is coated with carboxyl, epoxy group or toluene Huang acyl
Any one in base.Magnetic bead can influence accuracy using the endless bulk deposition of preceding solid phase, therefore should select good dispersion when selection,
It is few that magnetic bead number of uniting is placed for a long time, the slow magnetic bead of sinking speed.And using ferroso-ferric oxide as the magnetic bead of kernel, surface packet
The variety classes group hydrophily wrapped up in is preferable so that can effectively be detached, be washed between magnetic bead, settling flux, can dropped significantly
Low non-specific adsorption can greatly improve precision when reaction is really achieved equilibrium condition.Preferably, it is coated with cortisol antibody
Magnetic bead in, according to 1~2% ratio be coated with cortisol antibody.
Specifically, the cortisol BSA compounds of acridinium ester label of the present invention, wherein acridinium ester is multiple with cortisol BSA
The molar ratio of substance markers is closed between 1:2.5-1:Between 10, preferred molar ratio example is 1:5.Select good hydrophilic property (non-specific
Adsorb small) acridinium ester, the non-specific adsorption between free acridinium ester and solid-phase magnetic beads can be reduced, can be with by chemical modification
The hydrophobicity for changing acridinium ester achievees the purpose that reduce non-specific adsorption.Preferably, the skin of acridinium ester label of the present invention
Matter alcohol BSA compounds are Cortisol-3-BSA compounds.
Specifically, the cortisol calibration object is 0.08 μ for the alcohol hemocrinia cortisol antigen concentration diluted clearly that decorticates
The solution of g/L-6 μ g/L.
Specifically, the luminous cleaning solution A includes following components：The disodium hydrogen phosphate of 50-100g/L, the phosphorus of 5-30g/L
Acid dihydride sodium, the sodium chloride of 50-200g/L, 0.1%-10% Tween-20,0.1%-10% liquid bio preservative
The hydrogen peroxide of Proclin-300,0.5%-5% and the nitric acid of 1-2M, percentage composition are mass percentage.For example, above-mentioned
The cleaning solution A that shines may include following components：The chlorination of the disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L, 180g/L
Sodium, 10% Tween-20,2% liquid bio preservative Proclin-300,4% hydrogen peroxide and the nitric acid of 1M, percentage
Content is mass percentage.
Specifically, the luminous cleaning solution B includes following components：The disodium hydrogen phosphate of 50-100g/L, the phosphorus of 5-30g/L
Acid dihydride sodium, the sodium chloride of 50-200g/L, 0.1%-10% Tween-20,0.1%-10% liquid bio preservative
The sodium hydroxide and 0.1%-2%TritonX-100 of Proclin-300,0.01-2.5M, percentage composition are mass percentage.
For example, above-mentioned luminous cleaning solution B may include following components：The disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L,
The sodium chloride of 180g/L, 10% Tween-20,2% liquid bio preservative Proclin-300,1.5M sodium hydroxide and
1%TritonX-100, percentage composition are mass percentage.
Below by embodiment, the present invention is described in detail.
Embodiment
1, prepared by the Myoglobin reagent of coating cortisol antibody
1) it is, 2.0 μm of magnetic beads by 5mg grain sizes, is resuspended with the 50mM pH MES buffer solutions for being 6.0 ± 0.1, is shaken using whirlpool
Device mixing is swung, (notes centrifuge tube after fully shaking：Centrifuge tube lid not have magnetic bead residual) it is placed on Magneto separate frame, Zhi Daoshang
Clear liquid is completely thorough, is carefully moved with micropipettor and abandons supernatant, and repetitive operation is twice.
2), magnetic bead is resuspended to target volume in the MES buffer solutions for being 6.0 ± 0.1 with 50mM pH, and vortex oscillator mixing is filled
Divide after concussion and appropriate cortisol antibody is added according to 1.5% coating ratio, is mixed well rapidly using vortex oscillator, then added
Enter MES buffer solutions and the 5M ammonium sulphate buffers that 50mM pH are 6.0 ± 0.1, is mixed well rapidly using vortex oscillator,
Under the conditions of 37 DEG C coating is shaken up using vortex mixer overnight.
3), after reaction, centrifuge tube (centrifuge tube lid not have magnetic bead residual) is placed on Magneto separate frame, until upper
It after clear liquid is thorough, is carefully moved with micropipettor and abandons supernatant, 1mL confining liquids are added, the confining liquid is containing 5% (W/V) BSA's
The MES buffer solutions that 50mM pH are 6.0 ± 0.1,37 DEG C of conditions shake up closing 14h using vortex mixer.
4), centrifuge tube (centrifuge tube lid not have magnetic bead residual) is placed on Magneto separate frame, until supernatant is completely saturating
It after thorough, carefully moved with micropipettor and abandons supernatant, it is 7.4 ± 0.1 that the 10mmol pH containing 8% (w/v) BSA are used at 37 DEG C
PB buffer solution for cleaning liquid cleans magnetic bead, and centrifuge tube (centrifuge tube lid not have magnetic bead residual) is placed on by magnetic bead concentration 1mg/mL
On Magneto separate frame, after supernatant becomes completely thorough, is carefully moved with micropipettor and abandon supernatant, repeated washing 3 times, every time 1
Hour.
5) magnetic bead, is resuspended with containing 0.2% poloxamer reagent buffer, vortex oscillator mixes well, 2-8 DEG C of guarantor
It deposits.
2, prepared by the cortisol BSA compound liquid-phase reagents of acridinium ester label
1) the 20mM PBS acridinium ester purification buffers containing 2%BSA, are prepared with deionized water.
2) the cortisol BSA compounds of 50 μ g, are added, and 100 μ L acridinium ester purification buffers are added.
3) it is, 1 according to the molar ratio of acridinium ester and the compound substance markers of cortisol BSA:0.25-1 μ L are added in 5 ratio
Acridinium ester (2mg/mL).
4) it, is put into light culture case, 37 DEG C of shaking table mixings, light culture 2h.
5) blocking agent 1h, is added.
6), the cortisol BSA complex purifications for completing label.
7) 0.25-1 μ g/mL, by mass percentage, are diluted to 0.2% poloxamer reagent buffer marking fluid
Working solution.
3, prepared by the compound releasing agent of ANS salt
By mass percentage, with 5mg/ml ANS-Na (8-anilino-1-naphthalene sulfonic acid sodium salt), 0.1%NaN3、150mM
NaCl and 40mM barbital sodiums prepare the compound releasing agent of ANS salt, adjust pH to 7.0, are packed as one bottle per 100mL, are placed in 4 DEG C of guarantors
It deposits.
4, prepared by cortisol calibration object
1), the alcohol hormone serum that decorticates of 1000mL is placed in 37 DEG C of thermostat water baths, is inactivated.
2), by mass percentage, with 150mM NaCl, 1%Proclin-300 and 0.1%NaN3After fire extinguishing is added
It decorticates in alcohol hormone serum, is packed as one bottle per 100mL, is placed in 4 DEG C of preservations.
3), by the above-mentioned alcohol hormone serum buffer doubling dilution that decorticates prepared of cortisol antigen sterling:0.093,
0.214,0.493,1.134,2.609,6 μ g/L totally 6 bottles of calibration objects.
5, shine cleaning solution A, B preparation
1), shine cleaning solution A：The chlorination of the disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L, 180g/L is added
Sodium, 10% Tween-20,2% liquid bio preservative Proclin-300,4% hydrogen peroxide and the nitric acid of 1M, will be upper
It states substance to be sufficiently mixed, obtains the cleaning solution A that shines.
2), shine cleaning solution B：The chlorination of the disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L, 180g/L is added
The sodium hydroxide and 1%TritonX- of sodium, 10% Tween-20,2% liquid bio preservative Proclin-300,1.5M
100, above-mentioned substance is sufficiently mixed, the cleaning solution B that shines is obtained.
In efficient detection blood free cortisol kit by above-mentioned coating cortisol antibody Myoglobin reagent, acridine
The cortisol BSA compounds liquid-phase reagent of ester label, cortisol calibration object, the cleaning solution A that shines, shines at the compound releasing agent of ANS salt
Cleaning solution B compositions.
Project standard see the table below 1.
Table 1, project standard
Testing result is as follows：
1, the range of linearity：
With Logit-log models fittings, concentration range is 0-6 μ g/L, and the sample for concentration more than 6 μ g/L should be carried out first
It is measured again after dilution；Linear coefficient：R2≥0.99；As shown in the following table 2, attached drawing 1.
Table 2, Concentration Testing result statistical form
2. sensitivity for analysis:
It is detected with the matrix without measured object, replication 20 times, show that the RL Μ values of 20 testing results are (opposite
Luminous value), its average value (M) and standard deviation (SD) are calculated, the RL Μ values corresponding to M-2SD are obtained, according to without measured object
Concentration-RL Μ value results between matrix and low value calibration object carry out 2 regression fits and obtain linear function, and M-2SD institutes is right
The RL Μ values answered are brought into above-mentioned equation, and corresponding concentration value, as sensitivity for analysis are found out, and result should meet≤0.08 μ
G/L, data are as shown in table 3 below.
Table 3, sensitivity for analysis testing result statistical form
3, precision：
1) precision in, analyzing：2 mating quality-control products of test kit, i.e. low value quality-control product and high level quality-control product,
In same analysis, each quality-control product repeats detection 10 times, calculates the average value (M) and standard deviation (SD) of 10 measurement results, presses
Following formula (1) calculates the coefficient of variation (CV), and the test result coefficient of variation (CV) answers≤8.0%, and data are as shown in table 4 below.
2) precision between, analyzing：With 3 batch kits, 2 quality-control products in same set of kit are tested respectively, i.e.,
Low value quality-control product and high level quality-control product, each batch kit repeat to detect each sample 10 times, and 10 measurement results of calculating are put down
Mean value (M) and standard deviation (SD), as follows (1) calculate the coefficient of variation (CV), then between 3 lot number kits batch between become
Different coefficient (CV)≤15.0%, data are as shown in table 5 below.
Formula is as follows：
CV=SD/M × 100% ... ... ... ... (1)
In formula：CV-the coefficient of variation；
The standard deviation of SD-measurement result；
The average value of M-measurement result.
Precision testing result statistical form in table 4, analysis
| Precision in analysis | Quality-control product low value (μ g/L) | Quality-control product high level (μ g/L) |
| 1 | 0.310 | 2.903 |
| 2 | 0.309 | 2.976 |
| 3 | 0.306 | 2.900 |
| 4 | 0.301 | 2.929 |
| 5 | 0.316 | 2.826 |
| 6 | 0.312 | 3.109 |
| 7 | 0.323 | 2.853 |
| 8 | 0.302 | 3.050 |
| 9 | 0.324 | 3.020 |
| 10 | 0.310 | 2.887 |
| M | 0.311 | 2.950 |
| SD | 0.008 | 0.095 |
| CV | 2.5% | 3.2% |
Precision testing result statistical form between table 5, analysis
4, the rate of recovery：
Cortisol sample A of the concentration within the scope of 5 μ of μ g/L~6 g/L is added to concentration in 0.08 μ of μ g/L~0.2 g/L
In sample B in range, the volume ratio between the sample A being added and sample B is 1:9, it is calculated back according to following formula (2)
Yield R, as a result should meet rate of recovery 90%-110%, and test data is as shown in table 6 below.
In formula：The R-- rate of recovery；
The volume of V-- samples A；
V0The volume of-sample B；
The detectable concentration after sample A is added in c-sample B；
c0The detectable concentration of-sample B；
csThe concentration of-sample A.
Table 6, rate of recovery testing result statistical form
5. clinical test：
The present embodiment reagent compared with external authoritative reagent (Abbott Laboratories' reagent) 500 parts of clinical serum pattern detections as a result, surveying
Test result totally meets and reaches 99.9%, it is seen that kit prepared by the present embodiment method has preferable consistent with hospital's measured value
Property.Wherein 100 parts of clinical test results are enumerated, as shown in table 7 below.
Table 7, clinical test results statistical form
In conclusion the kit of a kind of compound releasing agent of ANS salt provided by the invention and detection Blood cortisol, this
The cortisol of combining form in blood of human body sample is changed into the cortex of free form by the compound releasing agent of ANS salt by invention
Alcohol, and pass through the content of chemiluminescent new technique detection free cortisol.The compound releasing agent of ANS salt of selection can be with difference
Glucocorticosteroid albumen is combined, and can fully release the cortisol of reference state in blood；The chemiluminescence
For acridinium ester chemiluminescent, acridinium ester chemiluminescent shines for flash type, and when illuminating is few, can be completely produced by catching reaction
Photon；Acridinium ester is artificial synthetic, higher to the anti-interference of sample interfering substance, is detected and is swum using acridinium ester label
It is very sensitive from the content of cortisol, keep the assay of total cortisol in blood sample more accurate.The present invention has operation
The advantages that simplicity, high sensitivity, verification and measurement ratio are fast, low-cost, convenient for automation.
It should be understood that the application of the present invention is not limited to the above for those of ordinary skills can
With improvement or transformation based on the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention
Protect range.
Claims (6)
1. a kind of kit of detection Blood cortisol, which is characterized in that including following components：It is coated with the magnetic of cortisol antibody
Pearl, the cortisol BSA compounds of acridinium ester label, the compound releasing agent of ANS salt and cortisol calibration object；
The molar ratio of acridinium ester and the compound substance markers of cortisol BSA is between 1:2.5-1:Between 10；
The cortisol BSA compounds of the acridinium ester label are Cortisol-3-BSA compounds；
The compound releasing agent of ANS salt includes following components：3-15mg/ml ANS salt, 0.1-0.5% NaN3、100-200mM
NaCl and 20-60mM barbital sodiums；
ANS salt has following structure described in the compound releasing agent of ANS salt：
；
Wherein R+For NH4 +、K+Or Na+；
The ANS salt directly dissolves in water.
2. the kit of detection Blood cortisol according to claim 1, which is characterized in that further include the cleaning solution that shines
A and luminous cleaning solution B.
3. the kit of detection Blood cortisol according to claim 1, which is characterized in that wrap on the surface of the magnetic bead
It is wrapped with any one in carboxyl, epoxy group, toluene Huang acyl group.
4. the kit of detection Blood cortisol according to claim 1, which is characterized in that the cortisol calibration
Product are the alcohol hemocrinia cortisol antigen diluted clearly that decorticates.
5. the kit of detection Blood cortisol according to claim 2, which is characterized in that the luminous cleaning solution A
Including following components：The disodium hydrogen phosphate of 50-100g/L, the sodium dihydrogen phosphate of 5-30g/L, 50-200g/L sodium chloride,
The hydrogen peroxide and 1- of liquid bio preservative Proclin-300,0.5%-5% of Tween-20,0.1%-10% of 0.1%-10%
The nitric acid of 2M.
6. the kit of detection Blood cortisol according to claim 2, which is characterized in that the luminous cleaning solution B
Including following components：The disodium hydrogen phosphate of 50-100g/L, the sodium dihydrogen phosphate of 5-30g/L, 50-200g/L sodium chloride,
The sodium hydroxide of liquid bio preservative Proclin-300,0.01-2.5M of Tween-20,0.1%-10% of 0.1%-10% and
0.1%-2% TritonX-100。
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| CN107290526A (en) * | 2017-08-24 | 2017-10-24 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of tetracycline and preparation method thereof |
| CN108982835A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of estradiol magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN109438539B (en) * | 2018-10-25 | 2020-07-14 | 美康生物科技股份有限公司 | Aldosterone derivative, chemiluminescence immunoassay kit containing aldosterone derivative and preparation method of aldosterone derivative |
| CN109444408A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of kit detecting animal blood cortisol |
| CN110579615A (en) * | 2019-09-06 | 2019-12-17 | 广州科方生物技术股份有限公司 | Release agent universal for steroid hormone in serum and preparation method thereof |
| CN110904059B (en) * | 2019-11-18 | 2021-03-23 | 华中农业大学 | Anthocyanin synthetase epitope peptide, antibody and application thereof |
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